Biodegradable polyphosphoramidates for controlled release of bioactive substances

ABSTRACT

The present invention is directed to a series of new polycationic biodegradable polyphosphoramidates. Process for making the polymers, compositions containing these polymers and bioactive ligands to enhance the cellular uptake ad intracellular trafficking, articles and methods for delivery of drugs and genes using these polymers are described. A gene delivery system based on these polymers is prepared by complex coacervation of nucleic acid (DNA or RNA) with polymers. Targeting ligands and molecules that could facilitate gene transfer can be conjugated to polymers to achieve selective and enhanced gene delivery. The current invention also provides a complex composition with buffering capacity.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/290,833 filed May 14, 2001, the teachings of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to biodegradable polymercompositions, in particular those containing both phosphoester linkagesin the polymer backbone and chargeable groups linked to the backbonethrough a P—N bond. The polymers of the invention are useful for drugand gene delivery, particularly as carriers for gene therapy and for thedelivery of protein drugs.

2. Background

Gene therapy has been progressively developed with the hope that it willbe an integral part of medical modalities in the future. Gene deliverysystem is one of the key components in gene medicine, which directs thegene expression plasmids to the specific locations within the body. Thecontrol of gene expression is achieved by influencing the distributionand stability of plasmids in vivo and the access of the plasmids to thetarget cells, and affecting the intracellular trafficking steps of theplasmids (Mahato, et al., 1999, Pharmaceutical perspectives of nonviralgene therapy, Adv. Genet. 41: 95-156). An ideal gene delivery carriershould be bioabsorable, non-toxic, non-immunogenic, stable duringstorage and after administration, able to access target cells, andefficient in aiding gene expression. As many studies demonstrated, thelimitations of viral vectors make synthetic vectors an attractivealternative. Advantages of non-viral vectors include non-immunogenicity,low acute toxicity, versatility, reproducibility and feasibility to beproduced on a large scale. Cationic liposome and cationic polymers arethe two major types of non-viral gene delivery carriers. Cationic lipidsself assemble into organized structures include micelles, plannarbilayer sheets, and lamellar vesicles. Through the condensation process,liposomes and cationic polymers form complexes with DNA due to chargeinteraction. A large variety of liposomal compositions have beendeveloped for gene delivery (Chesnoy and Huang, 2000. Structure andfunction of lipid-DNA complexes for gene delivery, Annu. Rev. Biophys.Biomol. Struct. 29: 27-47). An effective liposome vector generallycomposed of a positively charged lipid (e.g. cationic derivatives ofcholesterol and diacyl glycerol, quaternary ammonium detergents, lipidderivatives of polyamines, etc.) and a neutral helper lipid (e.g.dioleoyl phosphatidylethanolamine (DOPE) or dioleoyl phosphotidylcholine(DOPC)). Despite early excitement, there are serious limitations to mostcationic lipid systems. Several observations have suggested thatliposomal systems are relatively unstable after the administration.Significant toxicity upon repeated use has been shown to be associatedto liposomal vectors, especially the fusogenic phospholipid (neutrallipid), include the down regulation of PKC dependent immunomodulatorsynthesis, macrophage toxicity, neurotoxicity and acute pulmonaryinflammation (Filion and Phillips, 1998, Major limitations in the use ofcationic liposomes for DNA delivery, Int. J. Pharm. 162: 159-170).

Because of the limitations of viral vectors, cationic lipids, cationicpolymers as the basis of gene delivery systems have gained increasingattention recently. A number of polycations have been reported to effecttransfection of DNA, including poly-L-lysine, poly-L-ornithine,poly(4-hydroxy-L-proline ester), polyiminocarbonate containingcyclodextrin, poly[α-(4-aminobutyl)-L-glycolic acid], polyamidoamines,polyamidoamine dendrimers, chitosan, polyethylenimine,poly(2-dimethylaminoethyl methacrylate), etc. Significant progress hasbeen made in the development of polymer based systems, especiallybiodegradable polymers that have lower toxicity and can mediate genetransfection via condensing DNA into small particles and protecting DNAfrom enzymatic degradation. Nevertheless, searching for a safer and moreefficient gene carrier still remains a major challenge in the field ofnon-viral gene delivery.

SUMMARY OF THEM INVENTION

The invention provides positively chargeable biodegradable polymers thatcomprises at least one phosphoester linkage in the polymer backbone andat least one positively chargeable group wherein the positivelychargeable group is a substitutent of a side chain attached to thepolymer backbone through a phosphoramidate linkage, e.g., a P—N bond.

The invention further provides positively chargeable biodegradablepolymer compositions comprising:

-   -   (a) at least one biologically active substance; and    -   (b) A positively chargeable biodegradable polymer comprising at        least one phosphoester linkage in the polymer backbone and at        least one positively chargeable group wherein the positively        chargeable group is a substituent of a side chain attached to        the polymer backbone through a phosphoramidate linkage.

The invention additionally provides a method of preparing a positivelychargeable biodegradable polymers. The method comprising the steps of:

-   -   polymerizing at least two monomers to form a polymer with at        least one phosphoester linkage in the polymer backbone;    -   reacting the polymer with a primary or secondary amine having a        positively chargeable group or a substituent that can be        functionalized to a positively chargeable group under conditions        conducive to the formation of a positively chargeable        biodegradable polymer comprising at least one phosphoester        linkage in the polymer backbone and at least one positively        chargeable group wherein the positively chargeable group is a        substitutent of a side chain attached to the polymer backbone        through a phosphoramidate linkage.

The invention provides a method of preparing a positively chargeablebiodegradable polymer composition. The method comprises the steps of:

-   -   providing a positively chargeable biodegradable polymer        comprising at least one phosphoester linkage in the polymer        backbone and at least one positively chargeable group wherein        the positively chargeable group is a substitutent of a side        chain attached to the polymer backbone through a phosphoramidate        linkage.    -   contacting the positively chargeable biodegradable polymer with        a biologically active substance under conditions conducive to        the formation of a complex, e.g., a composition, comprising the        positively chargeable biodegradable polymer and the biologically        active substance.

The invention also provides for the controlled release of a biologicallyactive substance. The method comprises the steps of:

-   -   providing a positively chargeable biodegradable polymer        composition comprising:        -   (a) at least one biologically active substance; and        -   A positively chargeable biodegradable polymer comprising at            least one phosphoester linkage in the polymer backbone and            at least one positively chargeable group wherein the            positively chargeable group is a substituent of a side chain            attached to the polymer backbone through a phosphoramidate            linkage;    -   contacting the composition with a biological fluid, cell or        tissue under conditions conducive to the delivery of at least a        portion of the biologically active substance to the biological        fluid, cell or tissue.

The invention further provides methods for gene transfection using thecontrolled release methods and the positively chargeable biodegradablepolymer composition comprising a DNA sequence, a gene or a genefragment, to deliver a DNA sequence, a gene or a gene fragment to aspecified tissue target in a patient. Gene transfection methods of theinvention are suitable for use in treatment of any disease or disorderwhich is currently treatable by gene therapy or is contemplated as adisease or disorder suitable for treatment by gene therapy in the forfuture. Gene transfection methods of the invention comprise the steps of

-   -   providing a positively chargeable biodegradable polymer        composition comprising:        -   (a) at least one DNA fragment, gene or gene fragment; and        -   (b) a positively chargeable biodegradable polymer comprising            at least one phosphoester linkage in the polymer backbone            and at least one positively chargeable group wherein the            positively chargeable group is a substituent of a side chain            attached to the polymer backbone through a phosphoramidate            linkage;    -   contacting the composition with a biological fluid, cell or        tissue under conditions conducive to the delivery of at least a        portion of the DNA sequence gene or gene fragment to the        biological fluid, cell or tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Synthesis scheme of P5-SP;

FIG. 2. Gel permeation chromatograph of P5-SP;

FIG. 3. Structures of P5-SP, P5-BA, P5-DMA, P5-DEA and P5-TMA;

FIG. 4. Cytotoxicity of PPAs in COS-7 Cells as compared with PEI andPLL;

FIG. 5. Gel electrophoretic analysis of the complexation of PPAs withDNA;

FIG. 6. In vitro transfection efficiency of PPA-DNA coacervates in HEK293 cells;

FIG. 7. In vitro transfection efficiency of P5-SP-DNA coacervates in HEK293 cells at different charge ratios (+/−);

FIG. 8. Transfection of several cell lines using different polymericcarriers and PRE-Luciferase as a model plasmid. P5-SP-DNA coacervatesare tested with different charge ratios (5 and 10 for CaCo-2 cells, HeLacells and HUH 7 cells; 7.5 and 10 for HEK293 cells, COS-7 cells andHepG2 cells); and

FIG. 9. Transfection mediated by PPA-SP/PPA-DMA mixtures at differentmolar ratios in COS-7 cells and HeLa cells compared with PPA-SP andPPA-DMA alone.

DETAILED DESCRIPTION OF THE INVENTION

This invention discloses a new class of cationic biodegradable polymerscontaining phosphoester group in the backbone and chargeable groupslinked to the backbone through a phosphoramidate linkage, e.g., a P—Nbond. The biodegradable polyphosphoramidate of the invention comprisethe recurring monomeric units shown in the Formula I:

wherein:

R₁ is a divalent organic moiety that is aliphatic, aromatic orheterocyclic;

R₂ and R₃ are each independently selected from the group consisting ofhydrogen, alkyl, aryl, heteroaryl, heteroalicyclic, cycloalkyl, aralkylor cycloalkylalkyl;

each non-hydrogen occurrence of R₂ and R₃ is substituted with one ormore positively chargeable functional groups (e.g. primary amino group,secondary amino group, tertiary amino group and quaternary amino group,etc.); and

n is 5 to 2000.

Particularly preferred polymers according to formula I include polymersof formula II:

wherein:

n, R₂ and R₃ are as defined in Formula I;

R₄ and R₅ are independently selected from the group consisting ofhydrogen, alkyl, cycloalkyl, alkoxy, aryl, heteroaryl, heteroalicyclic,aralkyl, a steroid derivative; and

q is an integer from about 1 to about 5.

Preferred positively chargeable biodegradable polymers of the inventiona capable of forming a complex with biologically active substance.Preferred biologically active substances include DNA, RNA, proteins,small molecule therapeutics, and the like.

Other preferred positively chargeable biodegradable polymers of theinvention include polymers capable of complexing 20-60% by weight of abiologically active substance such as DNA, RNA, proteins, small moleculetherapeutics, and the like.

Furthermore, preferred positively chargeable biodegradable polymers ofthe invention include polymers having between about 5 and about 2,000phosphoramidate groups, more preferably between about 10 and about 1500phosphoramidate groups, and particularly preferred are polymers havingbetween about 20 and 1000 phosphoramidate groups. Also preferred arepolymers having a molecular weight of between about 1000 and 500,000,more preferable having a molecular weight of between about 2000 and200,000, particularly preferable are polymers having a molecular weightof between about 2000 and 100,000.

In additional preferred embodiments, positively chargeable biodegradablepolymers of the invention further comprise one or more groups thatfacilitate intracellular or extracellular delivery of a biologicallyactive substance. Preferred groups for facilitating intracellulardelivery of a biologically active substance include a lysosomalyticagent, an amphiphilic peptide, a steroid derivative, and the like.

In preferred embodiments, the biodegradable polyphosphoramidate polymersof the invention, including polymers according to Formula I or FormulaII, are biocompatible before and upon degradation.

In preferred embodiments, the biologically active substance isnegatively charged Preferred biologically active substances includeanionic groups such as phosphate groups, carboxylate groups, sulfategroups and other negatively charged bio-compatible groups.

In another embodiment the invention features a coacervate system usefulfor the delivery of bioactive macromolecules comprising thebiodegradable polymer of Formula I.

In another embodiment, the invention features polymer conjugatescomprising polymers of Formula I and bioactive ligands that couldfacilitate cell uptake and intracellular trafficking steps.

In another embodiment of the invention coacervate systems useful fordelivery of nucleic acids (DNA or RNA) and/or protein drugs and comprisethe biodegradable polymer of Formula I or the above-described polymerconjugates are described.

In a further embodiment, the invention contemplates a process of makingpolymeric coacervates for delivery of protein drugs or nucleic acid.

This invention also describes a number of procedures for preparing thebiodegradable polymers described above.

The biodegradable polymers could be copolymers having one or severaldifferent monomeric recurring units described in Formula I.

A lipophilic moiety, e.g. a group bearing cholesterol structural orlipid, could be conjugated to the carriers to enhance the interactionbetween complexes and cell membrane therefore facilitates cell uptake.

An endolysosomalytic agent, e.g. an amphiphilic peptide, could beconjugated to the carriers to enhance the endosomal escape after celluptake step.

A nucleus localization signal could be conjugated to the carriers tofacilitate the nucleus translocation.

It is a discovery of the present invention that nucleic acid moleculesof various chain lengths can complex with these biodegradable polymersof Formula I in aqueous conditions to form coacervates or solidmicroparticles ranging from submicron to microns in size. Thesecoacervates containing nucleic acids, when appropriately targeted, cantransfect cells with phagocytic activity.

According to the present invention, other molecules, especially thosecarry charges and have relatively higher molecular weights could also beincorporated into the complexes/coacervates.

In a further embodiment, the invention contemplates a process of makingpolymeric coacervates for delivery of bioactive macromolecules.

In yet another embodiments the invention comprises articles comprisingone or several different polymers with structures shown in Formula I andbioactive substances, e.g. nucleic acids and other negatively chargedmacromolecules for sustained release of these bioactive substancesin-vivo and/or in-vitro. Additionally, the bioactive substances can bereleased in a controlled, sustained manner either an intracellular andextracellular manner.

In a still further embodiment, the invention contemplates a process forpreparing biodegradable polyphosphoramidates, which comprises a step ofreacting a polymer shown in Formula III, wherein X is a halogen and R¹is as defined in Formula I, with a primary or secondary amine having ageneral structure as R²R₃NH, wherein R₂ and R₃ are each independentlyselected from the group consisting of hydrogen, alkyl aryl, heteroaryl,heteroalicyclic, cycloalkyl, aralkyl or cycloalkylalkyl wherein eachnon-hydrogen occurrence of R₂ and R₃ is substituted with one or morepositively chargeable functional groups (e.g. primary amino group,secondary amino group, tertiary amino group and quaternary amino group,etc.).

In specific, embodiments, one or more charged groups that are present inR₂ or R₃ are capable of reacting with a P-halogen bond. Preferably, suchreactive positively chargeable groups are protected using standardorganic chemistry protecting group techniques to prevent reaction ofsuch groups with the P—X bond. The protected primary or secondary amine,R₂R₃NH, is then reacted with the polymer of Formula III where X is ahalogen. In particular preferred embodiments, reactive positivelychargeable groups include primary or secondary amine groups which areprotected using standard amine protection methodologies.

In other preferred embodiments, phosphoramidate polymers of theinvention can be prepared by formation of a P—N linkage by reacting apolymer of Formula II wherein X is hydrogen with a primary or secondaryamine in a polar aprotic solvent mixture such as DMF/CCl₄ to Scheme 1.

The biodegradable polymeric system described in the present inventionachieves sustained and localized delivery of one or more therapeuticagents to a designated biological tissue or site in a patient. Inparticular, the polymeric system of the invention achieve sustained andlocalized delivery of one or more genes in skeletal muscles orintradermally and achieve a higher gene transfer efficiencies than otherplasmid delivery systems currently under investigation. Thebiodegradable polymeric carriers described in the present inventionachieve gene transfer efficiencies in vitro and in vivo that aresuperior to other polycationic carriers currently under investigation.

The polyphosphoramidate carriers of the present invention typicallyoffer the following advantages over other biodegradable carriersdescribed in the literatures and patents.

Polyphosphoramidate polymers of the invention are biodegradable whereinthe polymers have a cleavable backbone, either hydrolytically orenzymatically. The two most effective polymeric carriers currentlyavailable, PEI and various dendrimeric materials, are not biodegradableand their fate, in vivo, after administration is still unclear.

Polyphosphoramidate polymers of the invention are biocompatible before,during and after biodegradation. Biodegradation breakdown products aretypically non-toxic. The polyphosphoramidate polymers of the inventionare less cytotoxic than poly-L-lysine, PEI and liposome compositions invitro. In a preferred embodiment, polymers of Formula I are degraded tophosphate, 1,2-propanediol and amines R²R³NH. By prudent selection ofthe side chains, the polymer potentially has minimal toxicity before andupon degradation.

Polyphosphoramidate polymers described in the present patent have highermolecule weight than most other biodegradable carriers reported in theliteratures whose number average molecular weights are in the range of3,000 to 9,000. The biodegradable polymers described here generally havenumber average molecular weights in the range of 10,000 to 500,000.Higher molecular weight of the polymeric carriers generally increasesthe binding capacity of the carriers such that the polymers of theinvention typically exhibit superior uptake of DNA and protein.

The structures of polyphosphoramidate polymers are tailorable to havevariable charged groups with different pKb, different charge density,molecular weight, hydrophilicity/hydrophobicity balance to optimize thetransfection activity of the carriers. An endolysosomalytic agent, e.g.an amphiphilic peptide, may be conjugated to the carriers to enhance theendosomal escape after cell uptake step. A lipophilic moiety, e.g. agroup bearing cholesterol structural or lipid, may be conjugated to thecarriers to enhance the interaction between complexes and cell membranetherefore facilitate cell uptake. A nucleus localization signal could beconjugated to the carriers to facilitate the nucleus translocation.

Polyphosphoramidates suitable for use in the invention may be modifiedto comprise one or more specific ligands conjugated to the side chain oras a side chain group to enhance the cellular uptake of one or morebioactive molecules (nucleic acids and proteins) dispersed in carrierpolymer and/or achieve tissue/cell specific delivery of the bioactivecargo.

Polyphosphoramidate polymers suitable for use in the methods of theinvention typically posses higher molecular weights than polymericcarriers disclosed in the art such that complexes/coacervates comprisingthe polyphosphoramidates of the invention are more stable than otherpolycationic materials with lower molecular weights.

Polyphosphoramidate polymers and compositions comprising at least onebioactive molecule and a polyphosphoramidate polymer are prepared byreproducible and easily scalable procedures.

An attractive coacervate delivery system requires a delicate balanceamong factors such as the simplicity of preparation, cost effectiveness,nucleic acids loading level, controlled release ability, storagestability, and immunogenicity of the components. The gene deliverysystem described here may offer advantages compared to other particulatedelivery systems, including the liposomal system. The problems ofinstability, low loading level, and controlled release ability arebetter resolved with these polymeric systems. Compared to othersynthetic polymeric systems, such as the extensively studiedpolylactic/polyglycolic copolymers, the mild conditions of coacervateformulation are appealing. Unlike the solvent evaporation and hot-melttechniques used to formulate synthetic polymeric coacervates, complexcoacervation requires neither contact with organic solvents nor heat. Itis also particularly suitable for encapsulating bio-macromolecules suchas nucleic acids and proteins not only through passive solvent capturingbut also by direct charge-charge interactions.

Targeting ligands can be directly bound to the surface of thecoacervates. Alternatively, such ligands can be conjugated to thepolymeric carriers to form molecular conjugates, which then complex withnucleic acids and/or proteins. Targeting ligands according to thepresent invention are any molecules, which bind to specific types ofcells in the body. These may be any types of molecules for which acellular receptor exists. Preferably the cellular receptors areexpressed on specific cell types only. Examples of targeting ligandsthat may be used are hormones, antibodies, cell-adhesion molecules,oligosaccharides, drugs, and neurotransmitters.

The method of the present invention involves a coacervation processdescribed in U.S. Pat. No. 5,972,707 (Roy, et al., 1999, Gene DeliverySystem) and U.S. Pat. No. 6,025,337 (Truong, et al., 2000, SolidMicroparticles for Gene Delivery). The process is optimized in thisinvention to best suit the complexation of nucleic acids andbiodegradable carriers of Formula I.

It is a discovery of the present invention that different polymers withdifferent charged groups, e.g. different amino groups with a wide rangeof acidity (pKb), could be included into one coacervate/complex systemfor the intracellular delivery. Such a system could offer bufferingcapacity similar to that of PEI.

Polyphosphoramidates suitable for use in the methods of the presentinvention include any and all different single pure isomers and mixturesof two or more isomers. The term isomer is intended to includediastereoisomers, enantiomers, regioisomers, structural isomers,rotational isomers, tautomers, and the like. For compounds which containone or more stereogenic centers, e.g., chiral compounds, the methods ofthe invention may be carried out with a enantiomerically enrichedcompound, a racemate, or a mixture of diastereomers. Preferredenantiomerically enriched compounds have an enantiomeric excess of 50%or more, more preferably the compound has an enantiomeric excess of 60%,70%, 80%, 90%, 95%, 98%, or 99% or more.

Polyphosphoramidates suitable for use in the methods of the presentinvention include any and all molecular weight distribution profiles,i.e., polymers having a M_(w), or M_(n) of between 1 and about 50, moretypically a M_(w), or M_(n) between about 1.2 and about 10. Moreover,polyphosphroamidates of the invention have a polydispersity index ofbetween about 1 and about 5.

As also discussed above, typical subjects for administration inaccordance with the invention are mammals, such as primates, especiallyhumans.

Biodegradable polymers differ from non-biodegradable polymers in thatthey can be degraded during in vivo therapy. This generally involvesbreaking down the polymer into its monomeric subunits. In principle, theultimate hydrolytic breakdown products of polymers suitable for use inthe methods of the present invention should be biocompatible, non-toxicand easily excreted from a patient's body. However, the intermediateoligomeric products of the hydrolysis may have different properties.Thus, toxicology of a biodegradable polymer intended for implantation orinjection, even one synthesized from apparently innocuous monomericstructures, is typically determined after one or more toxicity analyses.

The biodegradable polymer of the invention is preferably sufficientlypure to be biocompatible itself and remains biocompatible uponbiodegradation. “Biocompatible” is defined to mean that thebiodegradation products and/or the polymer itself are nontoxic andresult in only minimal tissue irritation when instilled in the bladderor transported or otherwise localized to other tissues within a patient.

It will be appreciated that the actual preferred amounts of therapeuticagent or other component used in a given composition will vary accordingto the therapeutic agent being utilized including the polymer systembeing employed, the mode of application, the particular site ofadministration, etc. Optimal administration rates for a given protocolof administration can be readily ascertained by those skilled in the artusing conventional dosage determination tests conducted with regard tothe foregoing guidelines.

As used herein, “alky” is intended to include branched, straight-chainand cyclic saturated aliphatic hydrocarbon groups including alkylene,having the specified number of carbon atoms. Examples of alkyl include,but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl, t-butyl, n-pentyl, and s-pentyl. Alkyl groups typically have 1to about 16 carbon atoms, more typically 1 to about 20 or 1 to about 12carbon atoms. Preferred alkyl groups are C₁-C₂₀ alkyl groups, morepreferred are C₁₋₁₂-alkyl and C₁₋₆-alkyl groups. Especially preferredalkyl groups are methyl, ethyl, and propyl.

As used herein, “heteroalkyl” is intended to include branched,straight-chain and cyclic saturated aliphatic hydrocarbon groupsincluding alkylene, having the specified number of carbon atoms and atleast one heteroatom, e.g., N, O or S. Heteroalkyl groups will typicallyhave between about 1 and about 20 carbon atoms and about 1 to about 8heteroatoms, preferably about 1 to about 12 carbon atoms and about 1 toabout 4 heteroatoms. Preferred heteroalkyl groups include the followinggroups. Preferred alkylthio groups include those groups leaving one ormore thioether linkages and from 1 to about 12 carbon atoms, morepreferably from 1 to about 8 carbon atoms, and still more preferablyfrom 1 to about 6 carbon atoms. Alylthio groups having 1, 2, 3, or 4carbon atoms are particularly preferred. Prefered alkylsulfinyl groupsinclude those groups having one or more sulfoxide (SO) groups and from 1to about 12 carbon atoms, more preferably from 1 to about 8 carbonatoms, and still more preferably from 1 to about 6 carbon atoms.Alkylsulfinyl groups having 1, 2, 3, or 4 carbon atoms are particularlypreferred. Preferred alkylsulfonyl groups include those groups havingone or more sulfonyl (SO₂) groups and from 1 to about 12 carbon atoms,more preferably from 1 to about 8 carbon atoms, and still morepreferably from 1 to about 6 carbon atoms. Alylsulfonyl groups having 1,2, 3, or 4 carbon atoms are particularly preferred. Preferred aminoalkylgroups include those groups having one or more primary, secondary and/ortertiary amine groups, and from 1 to about 12 carbon atoms, morepreferably from 1 to about 8 carbon atoms, and still more preferablyfrom 1 to about 6 carbon atoms. Aminoalkyl groups having 1, 2, 3, or 4carbon atoms are particularly preferred.

As used herein, “heteroalkenyl” is intended to include branched,straight-chain and cyclic saturated aliphatic hydrocarbon groupsincluding alkenylene, having the specified number of carbon atoms and atleast one heteroatom, e.g., N, O or S. Heteroalkenyl groups willtypically have between about 1 and about 20 carbon atoms and about 1 toabout 8 heteroatoms, preferably about 1 to about 12 carbon atoms andabout 1 to about 4 heteroatoms. Preferred heteroalkenyl groups includethe following groups. Preferred alkylthio groups include those groupshaving one or more thioether linkages and from 1 to about 12 carbonatoms, more preferably from 1 to about 8 carbon atoms, and still morepreferably from 1 to about 6 carbon atoms. Alkenylthio groups having 1,2, 3, or 4 carbon atoms are particularly preferred. Preferedalkenylsulfinyl groups include those groups having one or more sulfoxide(SO) groups and from 1 to about 12 carbon atoms, more preferably from 1to about 8 carbon atoms, and still more preferably from 1 to about 6carbon atoms. Alkenylsulfinyl groups having 1, 2, 3, or 4 carbon atomsare particularly preferred. Preferred alkenylsulfonyl groups includethose groups having one or more sulfonyl (SO₂) groups and from 1 toabout 12 carbon atoms, more preferably from 1 to about 8 carbon atoms,and still more preferably from 1 to about 6 carbon atoms.Alkenylsulfonyl groups having 1, 2, 3, or 4 carbon atoms areparticularly preferred. Preferred aminoalkenyl groups include thosegroups having one or more primary, secondary and/or tertiary aminegroups, and from 1 to about 12 carbon atoms, more preferably from 1 toabout 8 carbon atoms, and still more preferably from 1 to about 6 carbonatoms. Aminoalkenyl groups having 1, 2, 3, or 4 carbon atoms areparticularly preferred.

As used herein, “heteroalkynyl” is intended to include branched,straight-chain and cyclic saturated aliphatic hydrocarbon groupsincluding alkynylene, having the specified number of carbon atoms and atleast one heteroatom, e.g., N, O or S. Heteroalkynyl groups willtypically have between about 1 and about 20 carbon atoms and about 1 toabout 8 heteroatoms, preferably about 1 to about 12 carbon atoms andabout 1 to about 4 heteroatoms. Preferred heteroalkynyl groups includethe following groups. Preferred alkynylthio groups include those groupshaving one or more thioether linkages and from 1 to about 12 carbonatoms, more preferably from 1 to about 8 carbon atoms, and still morepreferably from 1 to about 6 carbon atoms. Alkynylthio groups having 1,2, 3, or 4 carbon atoms are particularly preferred. Preferredalkynylsulfinyl groups include those groups having one or more sulfoxide(SO) groups and from 1 to about 12 carbon atoms, more preferably from 1to about 8 carbon atoms, and still more preferably from 1 to about 6carbon atoms. Alkynylsulfinyl groups having 1, 2, 3, or 4 carbon atomsare particularly preferred. Preferred alkynylsulfonyl groups includethose groups having one or more sulfonyl (SO₂) groups and from 1 toabout 12 carbon atoms, more preferably from 1 to about 8 carbon atoms,and still more preferably from 1 to about 6 carbon atoms.Alkynylsulfonyl groups having 1, 2, 3, or 4 carbon atoms areparticularly preferred. Preferred aminoalkynyl groups include thosegroups having one or more primary, secondary and/or tertiary aminegroups, and from 1 to about 12 carbon atoms, more preferably from 1 toabout 8 carbon atoms, and still more preferably from 1 to about 6 carbonatoms. Aminoalkynyl groups having 1, 2, 3, or 4 carbon atoms areparticularly preferred.

As used herein, “cycloalkyl” is intended to include saturated ringgroups, having the specified number of carbon atoms, such ascyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. Cycloalkyl groupstypically will have 3 to about 8 ring members.

In the term “(C₃₋₆ cycloalkyl)C₁₋₄ alkyl”, as defined above, the pointof attachment is on the alkyl group. This term encompasses, but is notlimited to, cyclopropylmethyl, cyclohexylmethyl, cyclohexylmethyl.

As used here, “alkenyl” is intended to include hydrocarbon chains ofstraight, cyclic or branched configuration, including alkenylene, andone or more unsaturated carbon-carbon bonds which may occur in anystable point along the chain, such as ethenyl and propenyl. Alkenylgroups typically will have 2 to about 12 carbon atoms, more typically 2to about 12 carbon atoms.

As used herein, “alkynyl” is intended to include hydrocarbon chains ofstraight, cyclic or branched configuration, including alkynylene, andone or more triple carbon-carbon bonds which may occur in any stablepoint along the chain, such as ethynyl and propynyl. Alkynyl groupstypically will have 2 to about 20 carbon atoms, more typically 2 toabout 12 carbon atoms.

As used herein, “haloalkyl” is intended to include both branched andstraight-chain saturated aliphatic hydrocarbon groups having thespecified number of carbon atoms, substituted with 1 or more halogen(for example —C_(v)F_(w) where v=1 to 3 and w=1 to (2v+1). Examples ofhaloalkyl include, but are not limited to, trifluoromethyl,trichloromethyl, pentafluoroethyl, and pentachloroethyl. Typicalhaloalkyl groups will have 1 to about 16 carbon atoms, more typically 1to about 12 carbon atoms.

As used herein, “alkoxy” represents an alkyl group as defined above withthe indicated number of carbon atoms attached through an oxygen bridge.Examples of alkoxy include, but are not limited to, methoxy, ethoxy,n-propoxy, i-propoxy, n-butoxy, 2-butoxy, t-butoxy, n-pentoxy,2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy,3-hexoxy, and 3-methylpentoxy. Alkoxy groups typically have 1 to about16 carbon atoms, more typically 1 to about 12 carbon atoms.

“Prodrugs” are intended to include any covalently bonded carriers whichrelease the active parent drug according to formula I in vivo when suchprodrug is administered to a mammalian subject. Prodrugs of a compoundare prepared by modifying functional groups present in the drug compoundin such a way that the modifications are cleaved, either in routinemanipulation or in vivo, to the parent compound.

Combinations of substituents and/or variables are permissible only ifsuch combinations result in stable compounds. A stable compound orstable structure is meant to imply a compound that is sufficientlyrobust to survive isolation to a useful degree of purity from a reactionmixture, and formulation into an effective therapeutic agent.

As used herein, the term “aliphatic” refers to a linear, branched,cyclic alkane, alkene, or alkyne. Preferred aliphatic groups in thepoly(phosphoester-co-amide) polymer of the invention are linear orbranched and have from 1 to 20 carbon atoms.

As used herein, the term “aryl” refers to an unsaturated cyclic carboncompound with 4n+2 electrons where n is a non-negative integer, about5-18 aromatic ring atoms and about 1 to about 3 aromatic rings.

As used herein, the term “heterocyclic” refers to a saturated orunsaturated ring compound having one or more atoms other than carbon inthe ring, for example, nitrogen, oxygen or sulfur.

The polymers of the invention are usually characterized by a releaserate of the therapeutic agent in vivo that is controlled at least inpart as a function of hydrolysis of the phosphoester bond of the polymerduring biodegradation. Additionally, the therapeutic agent to bereleased may be conjugated to the sidechain of the phosphramidate repeatunit to form a pendant drug delivery system. Further, other factors arealso important.

The life of a biodegradable polymer in vivo also depends upon itsmolecular weight, crystallinity, biostability, and the degree ofcross-linking. In general, the greater the molecular weight, the higherthe degree of crystallinity, and the greater the biostability, theslower biodegradation will be.

The therapeutic agent of the invention can vary widely with the purposefor the composition. The agnet(s) may be described as a single entity ora combination of entities. The delivery system is designed to be usedwith therapeutic agents having high water-solubility as well as withthose having low water-solubility to produce a delivery system that hascontrolled release rates. The terms “therapeutic agent” and“biologically active substance” include without limitation, medicaments;vitamins; mineral supplements; substances used for the treatment,prevention, diagnosis, cure or mitigation of disease or illness; orsubstances which affect the structure or function of the body; orpro-drugs, which become biologically active or more active after theyhave been placed in a predetermined physiological environment.

Non-limiting examples of useful therapeutic agents; and biologicallyactive substances include the following expanded therapeutic categories:anabolic agents, antacids, anti-asthmatic agents, anti-cholesterolemicand anti-lipid agents, anti-coagulants, anti-convulsants,anti-diarrheals, anti-emetics, anti-spasmodic agents, anti-inflammatoryagents, anti-manic agents, anti-nauseants, anti-neoplastic agents,anti-obesity agents, anti-pyretic and analgesic agents, anti-spasmodicagents, anti-thrombotic agents, anti-uricemic agents, anti-anginalagents, antihistamines, anti-tussives, appetite suppressants,biologicals, cerebral dilators, coronary dilators, decongestants,diuretics, diagnostic agents, erythropoietic agents, expectorants,gastrointestinal sedatives, hyperglycemic agents, hypnotics,hypoglycemic agents, ion exchange resins, laxatives, mineralsupplements, mucolytic agents, neuromuscular drugs, peripheralvasodilators, psychotropics, sedatives, stimulants, thyroid andanti-thyroid agents, uterine relaxants, vitamins, antigenic materials,and prodrugs.

Specific examples of useful therapeutic agents and biologically activesubstances, i.e., bioactive molecules, from the above categoriesinclude: (a) anti-neoplastics such as androgen inhibitors,antimetabolites, cytotoxic agents, immunomoldulators; (b) anti-tussivessuch as dextromethorphan, dextromethorphan hydrobromide, noscapine,carbetapentane citrate, and chlophedianol hydrochloride; (c)antihistamines such as chlorpheniramine maleate, phenindamine tartrate,pyrilamine maleate, doxylamine succinate, and phenyltoloxamine citrate;(d) decongestants such as phenylephrine hydrochloride,phenylpropanolamine hydrochloride, pseudoephedrine hydrochloride, andephedrine; (e) various alkaloids such as codeine phosphate, codeinesulfate and morphine; (f) mineral supplements such as potassiumchloride, zinc chloride, calcium carbonates, magnesium oxide, and otheralkali metal and alkaline earth metal salts; (g) ion exchange resinssuch as cholestryramine; (h) anti-arrhythmics such asN-acctylprocainamide; (i) antipyretics and analgesics such asacetaninophen, aspirin and ibuprofen; (j) appetite suppressants such asphenyl-propanolamine hydrochloride or caffeine; (k) expectorants such asguaifenesin; (l) antacids such as aluminum hydroxide and magnesiumhydroxide; (m) biologicals such as peptides, polypeptides, proteins andamino acids, hormones, interferons or cytokines and other bioactivepeptidic compounds, such as hGH, tPA, calcitonin, ANF, EPO and insulin;(n) anti-infective agents such as anti-fungals, anti-virals, antisepticsand antibiotics; and (o) antigenic materials, partricularly those usefulin vaccine applications.

Preferably, the therapeutic agent or biologically active substance isselected from the group consisting of DNA, polysaccharides, growthfactors, hormones, anti-angiogenesis factors, interferons or cytokines,and pro-drugs. In a particularly preferred embodiment, the therapeuticagent is a DNA vaccine comprising a DNA sequence encoding an antigen, aDNA sequence encoding a cytokine or a mixture of DNA sequences encodingan antigen and a cytokine.

The therapeutic agents are used in amounts that are therapeuticallyeffective. While the effective amount of a therapeutic agent will dependon the particular material being used, amounts of the therapeutic agentfrom about 1% to about 65% have been easily incorporated into thepresent delivery systems while achieving controlled release. Lesseramounts may be used to achieve efficacious levels of treatment forcertain therapeutic agents.

In addition, the polymer composition of the invention can also comprisepolymer blends of the polymer of the invention with other biocompatiblepolymers, so long as they do not interfere undesirably with thebiodegradable characteristics of the composition. Blends of the polymerof the invention with such other polymers may offer even greaterflexibility in designing the precise release profile desired fortargeted drug delivery or the precise rate of biodegradability desiredfor structural implants such as for orthopedic applications. Examples ofsuch additional biocompatible polymers include other polycarbonates;polyesters; polyorthoesters; polyamides; polyurethanes;poly(iminocarbonates); and polyanhydrides.

As a drug delivery device, the polymer compositions of the inventionprovide a polymeric matrix capable of sequestering a biologically activesubstance and provide predictable, controlled delivery of the substance.The polymeric matrix then degrades to non-toxic residues.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination (i.e., otherdrugs being administered to the patient), the severity of the particulardisease undergoing therapy, and other factors, including the judgment ofthe prescribing medical practitioner.

A positively chargeable biodegradable polymer composition of theinvention also may be packaged together with instructions (i.e. written,such as a written sheet) for treatment of a disorder as disclosedherein, e.g. instruction for treatment of a subject that is susceptibleto or suffering from a disease or disorder which may be treated byadministration of a bioactive molecule e.g., therapeutic agent,dispersed in the positively chargeable biodegradable polymercomposition.

A positively chargeable biodegradable polymer composition of theinvention be administered parenterally, preferably in a sterilenon-toxic, pyrogen-free medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle. The term parenteral asused herein includes injections and the like, such as subcutaneous,intradermal, intravascular (e.g., intravenous), intramuscular,intrasternal, spinal, intrathecal, and like injection or infusiontechniques, with subcutaneous, intramuscular and intravascularinjections or infusions being preferred.

A positively chargeable biodegradable polymer composition of theinvention also may be packaged together with instructions (i.e. written,such as a written sheet) for treatment of a disorder as disclosedherein, e.g. instruction for treatment of a subject that is susceptibleto or suffering from inflammation, cellular injury disorders, or immunesystem disorders.

The following examples are illustrative of the invention. All documentsmentioned herein are incorporated herein by reference.

EXAMPLES

The following examples are of offered by way of illustration and are notintended to limit the invention in any manner.

Example 1

Synthesis and Characterization of Polyphosphoramidates

1.1 Synthesis of P5-SP (Structure Shown in FIG. 3)

The synthetic scheme of P5-SP is shown in FIG. 1.Poly(4methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane) is synthesizedaccording to the procedure described in the literature (Biela T, PenczekS, and Slomkowski S, 1982, Racemic and optimal activepoly(4-methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane): Synthesis andoxidation to the polyacids. Makromol. Chem. Rapid Commun. 3: 667-671).Briefly, 2-hydroxy-4-methyl-1,3,2-dioxaphospholane (58 g, 0.475 mol)[freshly prepared according to Lucas' method (Lucas H J, Mitchell F W,Jr., and Scully C N, 1950. Cyclic phosphites of some aliphatic glycols.J. Am. Chem. Soc. 72: 5491-5497) is polymerized in 200 ml of freshlydried CHCl₃ at room temperature for 48 hours. Polymerization isinitiated with triisobutylaluminum (1 wt %, 4 ml of 15% solution inheptane). The polymer is obtained by precipitation into anhydrousbenzene. This polymer become insoluble in chloroform afterprecipitation, but it is soluble in anhydrous DMF.

Poly(4-methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane) (1.094 g, 8.9 mmolP—H groups) is dissolved in anhydrous DMF (10 ml). To this solution isadded 5 ml of anhydrous CCl₄ and N¹,N⁸-bis(trifluroacetyl)spermidinetrifluroacetate (10.8 mmol, 4.9 g) in 10 ml of DMF using a syringe,followed by addition of 5 ml of anhydrous triethylamine under ice-waterbath. The reaction is performed at 0 C for 30 minutes then at roomtemperature overnight. The resulted solution is concentrated and productis obtained by precipitating in water followed by drying under vacuum.

The resulted polymer is suspended in 30 ml of concentrated ammoniasolution and the mixture is stirred at 60 C for 16 hours. The solutionis concentrated and dialyzed against water overnight using a dialysistubing with a MWCO of 7,500. P5-SP is obtained after lyophilizing thedialyzed solution. The structure of P5-SP is confirmed by proton-NMR:δ(ppm): 1.35-1.4 (d, 3H), 1.5-1.7 (4H), 1.75-1.95 (2II), 2.65-2.95 (4H),3.0-3.2 (4H), 3.75-4.15 (m, 2H), 4.35-4.65 (d, b, 1H). FIG. 2 shows atypical chromatograph by gel permeation chromatography analysis ofP5-SP. It is indicated that P5-SP synthesized has a weight averagemolecular weight of 4.58×10⁴, and number average molecular weight of3.14×10⁴ (Polydispersity=1.46).

1.2 Synthesis of P5-DMA (Structure Shown in FIG. 3)

To a solution of poly(4-methyl-2-oxo-2-hydro-1,3,2-dioxaphospholane) inanhydrous DMF cooled with ice-water bath, was added 5 mL of anhydrousCCl₄ and N,N-dimethylethylenediamine (20% excess to P—H) solution in,followed by addition of large excess of triethylamine. The reaction wasperformed at 0 C for 30 minutes and at room temperature overnight.P5-DMA was obtained by dialysis against water using a dialysis memberwith a MWCO of 2,000.

To the solution of P5-DMA (100 mg) in 5 ml of methanol was added CH₃I (1ml) and the mixture was allowed to stay at room temperature overnight.The resulted solution was concentrated and precipitated into ether.P5-TMA was obtained as yellowish power. The number average molecularweight of P5-TMA was 2.62×10⁴ as measured by GPC/LS/RI method.

P5-BA, P5-DEA and P5-TMA (Structures are shown in FIG. 3) aresynthesized according to a similar procedure.

Example 2

Assay for the Cytotoxicity of Polyphosphoramidates (PPAs)

Cytotoxicity of polyphosphoramidates (P5-SP, P5-BA, P5-DMA, P5-DEA andP5-TMA) in comparison with other potential gene carriers (poly-L-lysine(PLL) and polyethylenimine (PEI) is evaluated using the WST-1 dyereduction assay. COS-7 cells are seeded in a 96 well plate 24 hoursbefore the assay at the density of 5×10⁴ cells/well The cells areincubated 4 hours with 100 μl of DMEM medium complemented with 10% fetalbovine serum (FBS) containing various PPAs, or PLL or PEI at differentconcentrations ranging from 0 to 500 μg/ml. The medium in each well isreplaced with 100 μl of fresh complete medium and cells are cultured foran additional 20 hrs. Ten microliters of WST-1 reagent (Roche MolecularBiochemicals) is added to each well and allowed reacting for 4 hrs at37° C. The absorbance of the supernatant at 450 nm (use 655 nm as areference wavelength) is measured using a microplate reader (Model 550,Bio-Rad Lab. Hercules, Calif.).

The assay results (FIG. 4) indicate that polyphosphoramidates exhibitlower cytotoxicity in culture than widely used polycationic carrier, PLLand PEI. The LD₅₀ of PEI in this assay is 20 μg/ml, LD₅₀ of PLL is 42μg/ml, LD₅₀ of P5-SP is 85 μg/ml, LD₅₀ of P5-BA is 300 μg/ml, LD₅₀ ofDMA or DEA or TMA is well beyond 500 μg/ml. It is clear that PPAs havelower cytotoxicity than PLL and PEI. PPAs with tertiary amino group andquaternary amino groups have the lowest cytotoxicity in this assay.

Example 3

Gel Retardation Assay for the DNA Binding Capacity of PPAs

The formation of PPA-DNA coacervates is examined be theirelectrophorectic mobility on an agarose gel at various charge ratios ofPPAs to plasmid DNA (FIG. 5). No migration of the plasmid DNA occurredat charge ratio larger than 1.0 (P5-DMA, P5-DEA and P5-TMA) or 1.5(P5-BA) or 2.0 (P5-SP). This lack of migration is due to neutralizationof the nucleic acid by PPAs, suggesting the polycationic nature of PPAs.PPAs with tertiary amino groups and quternary amino groups appear tohave higher DNA binding capacity at the same charge ratio.

Example 4

Preparation of PPA-DNA Coacervates and Coacervates with ChloroquineSulfate

PPA is dissolved in saline at a concentration of 2-10 mg/ml. To thissolution is added plasmid DNA dissolved in saline to yield desired N/Pratios, followed by brief vortexing, and the mixture is allowed to standat room temperature for 30 minutes. The coacervates prepared accordingto this procedure are used directly for transfection study unless statedotherwise. The efficiency of complexation of DNA is close to 100% whenthe N/P ratio is over 1.0 as revealed by gel electrophoretic mobilityanalysis.

Chloroquine sulfate (CQ) has been widely proven to be an effectivereagent to disrupt lysosomes and enhance the transfection efficiency inmany polycationic gene delivery systems. CQ is co-encapsulated into thecoacervates simply by incorporating CQ into the PPA solution and theirforming coacervates according to the same procedure. The CQ incorporatedcoacervates are used for in vitro transfection without furtherpurification since the total amount of CQ added is still within thenon-toxic concentration range.

Example 5

Transfection Efficiency of PCEP-DNA Complex in Different Cell Lines

In vitro transfection of HEK 293 cells with PPA-DNA coacervates isevaluated using luciferase as a marker gene. Cells are seeded 24 hoursprior to transfection into a 24-well plate (Becton-Dickinson, LincolnPark, N.J.) at a density of 8×10⁴ per well with 1 ml of complete medium(DMEM containing 10% FBS, supplemented with 2 mM L-glutamate, 50units/ml penicillin and 50 μg/ml streptomycin). At the time oftransfection, the medium in each well is replaced with 1 ml of serumfree DMEM. PPA-DNA coacervates or PEI-DNA complexes or PLL-DNA complexesor Transfast™-DNA complexes are incubated with the cells for 3 hours at37° C. The medium is replaced with 1 ml of fresh complete medium andcells are further incubated for 48 hours. All the transfection tests areperformed in triplicate. After the incubation, cells are permeabilizedwith 200 μl of cell lysis buffer (Promega Co., Madison, Wis.). Theluciferase activity in cell extracts is measured using a luciferaseassay kit (Promega Co., Madison, Wis.) on a luminometer (Lumat9605, EG&GWallac). The light units (LU) are normalized against proteinconcentration in the cell extracts, which is measured using BCA proteinassay kit (Pierce, Rockford, Ill.).

FIG. 6 shows the transfection efficiency of PPA-DNA coacervates preparedfrom five different PPAs with 100 μg/ml of CQ or without CQ, comparingwith PEI, PLL and Transfast as gene carriers. Coacervates prepared withP5-SP in the presence of CQ result in the highest transfectionefficiency, similar to the level obtained by Transfast-DNA complexes andPEI-DNA complexes. Other PPAs only show moderate transfection activity.It is also evident that CQ can enhance the transfection efficiency forabout 4 times at a concentration of 40 μg/ml of CQ, transfectionefficiency increases with dose and peaks at a dose of 80 μg/ml of CQ(data not shown). The following experiments are performed with 100 μg/mlof CQ incorporated in the coacervates.

As the gel electrophoresis analysis shows, at a +/− charge ratio of 1.0(P5-DMA, P5-DEA and P5-TMA) or 1.5 (P5-BA) or 2.0 (P5-SP) and above, allthe plasmid DNA added to the preparation mixture is complexed with PPAs.Coacervates prepared at different charge ratios are also examined fortheir abilities to transfect HEK293 cells (FIG. 7). Although completeDNA incorporation occurs at charge ratio of 2.0 and above for P5-SP, thehighest level of gene transfection is observed when the coacervates aresynthesized at the +/− charge ratios between 7.5 and above, Transfectionefficiency slightly decreases when the charge ratio is 12.5 and above.

The transfection efficiency is measured against five other cell linesusing PPA-DNA coacervates containing pRE-Luciferase plasmid (FIG. 8).Like in HEK293 cells, the highest level of luciferase expression inCaCo-2 cells, HeLa cells, IIuH-7 cells, COS-7 cells and HepG2 cells isalso found to be at a +/− ratio between 5 and 10. The transfectionefficiency in CaCO-2 cells, HeLa cells or HuH 7 cells is about 100 to200 times higher than PLL mediated transfection, and 10 to 50 timeslower than that obtained with PHI-DNA complexes. Transfection efficiencyin COS-7 cells or HepG2 cells is about 100 to 300 times higher than PLLmediated transfection and 2 folds lower than that obtained with PEI-DNAcomplexes.

Example 6

Gene Transfection Mediated by PPA Mixtures

Complexes comprising plasmid DNA and PPA mixture were prepared accordingto a similar procedure as described in Example 4, except that PPA-SP andPPA-DMA were pre-mixed at different ratios before complexation withplasmid DNA. DNA-polymer complexes were formed by adding 50 μl ofpolymer solution containing varying amounts of polymer to 50 μl ofvortexing pRE-luciferase (60 μg/ml, in 0.9% NaCl, pH 7.4) and vortexedfor 15-30 s. Complexes were allowed to form for 30 min at roomtemperature. The complexes were, used for transfection study withoutfurther purification.

This is based on the hypothesis that complexes containing various typesof amino groups would increase the buffering capacity, thus improve theintracellular delivery of the DNA to cytosol and nucleus. Transfectionof COS-7 cells using PPA-SP (containing primary amino group), PPA-DMA(containing tertiary amino group) or PPA-SP/PPA-DMA mixture showed thatPPA-SP/PPA-DMA mixture mediated significantly higher levels of geneexpression than either polymer alone (FIG. 9, structures see FIG. 3).Transfection was performed with 3 μg DNA per well. The charge ratio oftotal positive charges in PPA to negative charges in DNA was maintainedat 9. Under optimal condition (at a PPA-SP/PPA-DMA molar ratio of 4 to9), transfection efficiency achieved by PPA-SP/PPA-DMA mixture was 20and 160 times higher than PPA-SP and PPA-DMA mediated transfection,respectively.

This method of introducing polymeric carriers with different chargedgroups into the same complexes represents a simple yet effectiveapproach in developing polymeric gene carriers and understanding themechanisms of polymer mediated gene transfer.

1. A water soluble and positively charged biodegradablepolyphosphoramidate that is capable of forming a complex with negativelycharged bioactive macromolecules in aqueous solutions and comprises therecurring monomeric unit shown in Formula I,

wherein R₁ is a divalent aliphatic organic moiety; R₂ and R₃ are eachindependently selected from the group consisting of hydrogen, alkyl, orheteroalicyclic groups; each non-hydrogen occurrence of R₂ and R₃ issubstituted with one or more positively charged groups; and n is from 20to 2,000.
 2. A positively charged biodegradable polyphosphoramidate ofclaim 1, wherein the biodegradable polyphosphoramidate has between about20 and about 2,000 phosphoramidate groups.
 3. A positively chargedbiodegradable polyphosphoramidate of claim 1, wherein non-hydrogenoccurrences R₂ and R₃ are substituted with one or more charged groupsselected from the group consisting of primary amine, secondary amine,tertiary amine, quaternary amine or imidazoyl.
 4. A positively chargedbiodegradable polyphosphoramidate of claim 2, wherein one or more of R₁,R₂ or R₃ is substituted with one or more groups capable of facilitatingintracellular delivery of a negatively charged bioactive macromolecules,selected from the group consisting of lysosomalytic agent, anamphiphilic peptide, or a steroid derivative.
 5. A positively chargedbiodegradable polyphosphoramidate of claim 4, wherein the group capableof facilitating intracellular delivery of negatively charged bioactivemacromolecules is a cholesteryl group.
 6. A positively chargedbiodegradable polyphosphoramidate of claim 1, wherein R₁ is defined inFormula II,

wherein each occurrence of R₃ and R₄ are independently selected from thegroup consisting of hydrogen or alkyl group; and q is 2 to
 4. 7. Apositively charged biodegradable polyphosphoramidate composition formedby complexation in aqueous solutions comprising: (a) at least onenegatively charged bioactive macromolecule; and (b) a water soluble andpositively charged biodegradable polyphosphoramidate of claim
 1. 8. Apositively charged biodegradable polyphosphoramidate composition ofclaim 7, wherein the negatively charged bioactive macromolecules areselected from the group consisting of DNA, RNA, proteins, andpolysaccharides.
 9. A positively charged biodegradablepolyphosphoramidate composition of any one of claims 7 and 8, whereinthe biodegradable polyphosphoramidate is capable of complexing 20-60% byweight of the negatively charged biomacromolecules.
 10. A method ofpreparing a water soluble and positively chargeable biodegradablepolyphosphoramidate of Formula I, comprising the steps of: (a) reactinga precursor polymer with recurring unit shown in Formula III,

wherein R₁ is a divalent aliphatic organic moiety; with a primary orsecondary amine having a structure of HNR₂R₃, wherein each occurrence ofR₂ and R₃ are selected from the group consisting of hydrogen orpositively charged alkyl or heteroalicyclic containing protected primaryamine, protected secondary amine, tertiary amine, and quaternary amine;followed by (b). deprotecting the protected amino groups, if applicable.11. A method of preparing a positively charged biodegradablepolyphosphoramidate of claim 10, wherein the biodegradablepolyphosphoramidate has between about 20 and about 200 phosphoramidategroups.
 12. A method of preparing a positively charged biodegradablepolyphosphoramidate composition of claim 7, comprising the steps of:mixing an aqueous solution of the positively charged biodegradablepolymer of Formula I with concentrations ranging from 1 μg/ml to 500μg/ml, with an aqueous solution of one or more biological activemacromolecules, which is able to complex with polymer of Formula I. 13.A method of preparing a positively charged biodegradablepolyphosphoramidate composition of claim 12, wherein the negativelycharged or bioactive macromolecules are selected from the groupconsisting of DNA, RNA, proteins, and polysaccharides.
 14. A method ofpreparing a positively charged biodegradable polyphosphoramidatecomposition of claim 12 or 13, wherein the biodegradablepolyphosphoramidate is capable of complexing 20-60% by weight of thenegatively charged bioactive macromolecules.
 15. A method of preparing apositively charged biodegradable polyphosphoramidate composition ofclaim 12 or 13, wherein the biodegradable polyphosphoramidate hasbetween about 20 and about 200 phosphoramidate groups.
 16. A method forthe controlled release of a bioactive macromolecule comprising the stepsof: providing a positively charged biodegradable polyphosphoramidatecomposition of claim 7, and contacting the composition in vivo or invitro with a biological fluid, cell or tissue under conditions conduciveto the delivery of at least a portion of the biologically activesubstance to the biological fluid, cell or tissue so that thebiologically active substance is released in a controlled manner.
 17. Amethod of claim 16, wherein the bioactive macromolecule is releasedin-vivo.
 18. A method of claim 16, wherein the bioactive macromoleculeis released in-vitro.
 19. A method of claim 16, wherein the bioactivemacromolecule is released extracellularly.
 20. A method of claim 16,wherein the bioactive macromolecule is released intracellularly.
 21. Amethod of claim 16, wherein the bioactive macromolecule(s) are selectedfrom the group consisting of DNA, RNA, proteins, and polysaccharides.22. A method of claim 16, wherein the biodegradable polymer is capableof complexing 20-60% by weight of the negatively charged bioactivemacromolecule.
 23. A method of claim 16, wherein the biodegradablepolymer has between about 20 and about 200 phosphate groups.
 24. Amethod of claim 16, wherein the bioactive macromolecule is a growthfactor.
 25. A method of claim 16, wherein the bioactive macromolecule isselected from the group consisting of DNA sequences, genes, genefragments, DNA encoding vaccines, therapeutic agents, cytokines,immunoadjuvants, cancer therapeutic agents, proteins, and combinationsthereof.
 26. A method of claim 25, wherein DNA sequence, gene or genefragment is administered in connection with gene therapy.
 27. A methodof any one of claims 17 through 26 wherein the positively chargedbiodegradable polyphosphoramidate composition, including complexes ornanoparticles is delivered in vivo.